IDO inhibitors and methods of use

ABSTRACT

Compounds, compositions and methods for the treatment of malignancy are disclosed.

This application is a continuation of U.S. patent application Ser. No.10/550,444, filed Jun. 1, 2006 now U.S. Pat. No. 7,714,139, which is a35 USC §371 application based on PCT/US2004/005154, filed Feb. 20, 2004,which claims priority under 35 U.S.C. §119(e) to U.S. ProvisionalApplication No. 60/527,449, filed on Dec. 5, 2003, and U.S. ProvisionalApplication No. 60/458,162, filed on Mar. 27, 2003, the entire contentsof all of the aforementioned applications are incorporated by referenceherein.

FIELD OF THE INVENTION

This invention relates to the field of oncology. Specifically, theinvention provides novel chemotherapeutic agents and methods of usingsuch agents for the treatment of cancer.

BACKGROUND OF THE INVENTION

Tumors characteristically express atypical, potentially immunoreactiveantigens that are collectively referred to as tumor antigens.Accumulating evidence suggests that the failure of the immune system tomount an effective response against progressively growing tumors is notattributable to a lack of recognizable tumor antigens. Immunosuppressionby tumors is poorly understood and mechanisms by which tumors may escapeimmune surveillance have been poorly explored. Recently, it has beenshown that cytotoxic T cells become tolerized by a reduction in localconcentrations of tryptophan that are elicited by indoleamine2,3-dioxygenase (IDO) activity.

IDO is an oxidoreductase that catalyzes the rate-limiting step intryptophan catabolism. This enzyme is structurally distinct fromtryptophan dioxygenase (TDO), which is responsible for dietarytryptophan catabolism in the liver. IDO is an IFN-γ target gene that hasbeen suggested to play a role in immunomodulation (Mellor and Munn(1999) Immunol. Today, 20:469-473). Elevation of IDO activity depletesthe levels of tryptophan in local cellular environments. Induction ofIDO in antigen-presenting cells, where IDO is regulated by IFN-γ, blocksthe activation of T cells, which are especially sensitive to tryptophandepletion. T cells must undergo 1-2 rounds of cell division to becomeactivated, but in response to tryptophan depletion they arrest in G1instead. In this way, IDO has been proposed to inhibit the T_(H)1responses that promote cytotoxic T cell development.

The main evidence for the role of IDO in immunosuppression isdemonstrated by the ability of 1-methyl-tryptophan (1MT), a specific andbioactive IDO inhibitor (Cady and Sono (1991) Arch. Biochem. Biophys.291:326-333), to elicit MHC-restricted and T cell-mediated rejection ofallogeneic mouse concepti (Mellor et al. (2001) Nat. Immunol. 2:64-68;Munn et al. (1998) Science. 281: 1191-93). This effect is consistentwith the high levels of IDO expression in placental trophoblast cells(Sedlmayr et al. (2002) Mol. Hum. Reprod. 8:385-391).

Significantly, IDO activity has been shown to be elevated frequently inhuman tumors and/or in cancer patients (Yasui et al. (1986) Proc. Natl.Acad. Sci. USA. 83:6622-26; Taylor and Feng (1991) FASEB J. 5:2516-22).Since IDO can modulate immune responses, one logical implication is thatIDO elevation in cancer may promote tumor immunosuppression (Mellor andMunn (1999) Immunol. Today, 20:469-473; Munn et al. (1999) J. Exp. Med.189:1363-72; Munn et al. (1998) Science. 281:1191-93). This possibilityis supported by the observation that many cancers, including breastcancer, are characterized by a loss of beneficial immune functions thatcan limit malignant development. For example, T_(H)1 responses (of whichIFN-γ production is a hallmark) that promote the production of cytotoxicT cells are suppressed during cancer progression. A resultant hypothesisfrom this data was that if IDO drives cancer progression by blunting Tcell activation, then IDO inhibition in animals should blunt tumorgrowth by reversing IDO-mediated immunosuppression. However, delivery ofthe IDO inhibitor 1-methyl-tryptophan (1MT) only retarded and did notprevent tumor growth in a mouse model (Friberg et al. (2002) Int. J.Cancer 101:151-155; U.S. Pat. No. 6,482,416).

Cellular signal transduction, i.e., the series of events leading fromextracellular events to intracellular sequalae, is an aspect of cellularfunction in both normal and disease states. Numerous proteins thatfunction as signal transducing molecules have been identified, includingreceptor and non-receptor tyrosine kinases, phosphatases and othermolecules with enzymatic or regulatory activities. These moleculesgenerally demonstrate the capacity to associate specifically with otherproteins to form a signaling complex that can alter cellularproliferation.

Aberrant signal transduction can lead to malignant transformation,growth, and progression. Accordingly, inhibitors of signal transductionpathways have been used to treat cancer. During the past few years, anumber of signal transduction inhibitors (STIs) have been developed andtheir ability to suppress tumor growth is currently under investigation.

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, novel inhibitors ofindoleamine 2,3-dioxygenase (IDO) activity are provided. The novelcompounds have a formula selected from the group consisting of formula(I):

wherein R₁ is H or lower alkyl; R₂ is H; R₃ is selected from the groupconsisting of: (a)

wherein R_(A) and R_(B) are independently selected from the group of Hand hydrocarbyl;

wherein R_(C) is selected from the group of H and hydrocarbyl;

wherein n is a whole number from 1 to 10 and R_(D) is a carbolinesubstituent of the formula:

wherein R_(A) and R_(B) are independently selected from the group of Hand hydrocarbyl; or R₂ and R₃ are joined together and represent part ofa ring which is fused to the pyrrole moiety of formula (I) and which isselected from the group of:

wherein R_(E) is a hydrocarbyl or alkyl-Q, Q representing a substituentof the formula:

the compound of formula (I) being a β-carboline derivative when R₂ andR₃ joined together represent (i), a brassilexin derivative when R₂ andR₃ joined together represent (ii), and an N-substituted brassilexinderivative when R₂ and R₃ joined together represent (iii); X, Y, and Zmay be the same or different and are selected from the group consistingof H, halogen, NO₂, and hydrocarbyl; and when R₂ and R₃ are joinedtogether and represent part of a ring system, Y may also beisothiocyanate; with the proviso that formula (I) does not include anyof the compounds: 3-(N-methyl-thiohydantoin)-indole,3-(N-phenyl-thiohydantoin)-indole, 3-(N-allyl-thiohydantoin)-indole,5-methyl-brassinin, brassinin, brassilexin, β-carboline,3-butyl-β-carboline, 3-butyl-β-carboline,6-fluoro-3-carbomethoxy-β-carboline,6-isothiocyanate-3-carbomethoxy-β-carboline, 3-propoxy-β-carboline,3-carboxy-β-carboline, 3-carbopropoxy-β-carboline,3-carbo-tert-butoxy-β-carboline; and formula (II):

wherein X, Y, and Z may be the same or different and are selected fromthe group consisting of H, halogen, NO₂, and hydrocarbyl; and with theproviso that formula (II) does not include 3-amino-2-naphthoic acid.

According to another aspect of the present invention, a method fortreating cancer in a patient is provided. The method comprisesadministering an effective amount of a pharmaceutical compositioncomprising at least one indoleamine 2,3-dioxygenase (IDO) inhibitor,preferably a novel inhibitor of the instant invention, in apharmaceutically acceptable carrier medium.

In another embodiment of the invention, a method for treating cancer ina patient in need thereof is provided. The method comprisesadministering to the patient, concurrently or sequentially, an effectiveamount of at least one indoleamine 2,3-dioxygenase (IDO) inhibitor andat least one signal transduction inhibitor (STI). In a particularembodiment of the invention, the at least one STI is selected from thegroup consisting of bcr/abl kinase inhibitors, epidermal growth factor(EGF) receptor inhibitors, her-2/neu receptor inhibitors, and farnesyltransferase inhibitors (FTIs). The compounds may be administered in apharmaceutically acceptable carrier medium.

In still another embodiment of the invention, another method fortreating cancer in a patient in need thereof is provided. The methodcomprises administering to the patient, concurrently or sequentially, aneffective amount of at least one indoleamine 2,3-dioxygenase (IDO)inhibitor and at least one chemotherapeutic agent. In a particularembodiment of the invention, the at least one chemotherapeutic agent isselected from the group consisting of paclitaxel) (Taxol®, cisplatin,docetaxol, carboplatin, vincristine, vinblastine, methotrexate,cyclophosphamide, CPT-11, 5-fluorouracil (5-FU), gemcitabine,estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenictrioxide, irinotecan, and epothilone derivatives. The compounds may beadministered in a pharmaceutically acceptable carrier medium.

According to yet another aspect of the instant invention, a method isprovided for treating cancer in a patient in need thereof byadministering to the patient, concurrently or sequentially, an effectiveamount of at least one immunomodulator other than an IDO inhibitor andan effective amount of at least one cytotoxic chemotherapeutic agent orat least one STI. In a particular embodiment the at least oneimmunomodulator is selected from the group consisting of CD40L, B7,B7RP1, ant-CD40, anti-CD38, anti-ICOS, 4-IBB ligand, dendritic cellcancer vaccine, IL2, IL12, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18,TNF, IL-15, MDC, IFNa/b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10. Inanother particular embodiment, the at least one cytotoxicchemotherapeutic agent is selected from the group consisting ofpaclitaxel (Taxol®), cisplatin, docetaxol, carboplatin, vincristine,vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil(5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin),etoposide, arsenic trioxide, irinotecan, and epothilone derivatives.

In yet another embodiment of the present invention, a method is providedfor treating a chronic viral infection in a patient in need thereof byadministering to the patient, concurrently or sequentially, an effectiveamount of at least one indoleamine 2,3-dioxygenase (IDO) inhibitor andat least one chemotherapeutic agent. The at least one chemotherapeuticagent may be selected from the group consisting of paclitaxel (Taxol®),cisplatin, docetaxol, carboplatin, vincristine, vinblastine,methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5-FU),gemcitabine, estramustine, carmustine, adriamycin (doxorubicin),etoposide, arsenic trioxide, irinotecan, and epothilone derivatives. Theactive agents may be administered with or without a pharmaceuticallyacceptable carrier medium. The method of the invention may be used totreat chronic viral infection selected from the group consisting of:hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus(CMV), Epstein-Barr virus (EBV), varicella zoster virus, coxsackievirus, human immunodeficiency virus (HIV).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a scheme for synthesizing thiohydantoin derivatives ofthe present invention.

FIG. 2 provides a scheme of the present invention for derivatizingindole at C-4 and N-1 positions.

FIG. 3 provides a scheme for synthesizing C-6 substituted indoles of thepresent invention.

FIG. 4 provides a scheme for synthesizing C-4, C-5, and C-6trisubstituted indoles of the present invention.

FIG. 5 provides an alternative synthesis scheme of thiohydantoinderivatives of the present invention.

FIG. 6 provides a scheme for synthesizing brassinin derivatives of thepresent invention.

FIG. 7 provides a scheme for synthesizing C-5 and C-6 substitutedβ-carboline derivatives of the present invention.

FIG. 8 provides a scheme for synthesizing C-3 alkyl substitutedβ-carboline derivatives of the present invention.

FIG. 9 provides an alternative synthesis scheme of β-carbolinederivatives of the present invention.

FIG. 10 provides a scheme for synthesizing 3-amino-6/7-bromo-2-naphthoicacid of the present invention.

FIG. 11 provides a scheme for synthesizing C-6, C7, and C-8trisubstituted 3-amino-2-naphthoic acids of the present invention.

FIG. 12 provides an alternative synthesis scheme of C-6, C-7, and C-8trisubstituted 3-amino-2-naphthoic acids of the present invention.

FIG. 13 provides a scheme for synthesizing brassilexin derivatives ofthe present invention.

FIG. 14 provides an alternative scheme for synthesizing substitutedbrassilexin derivatives of the present invention.

FIG. 15 provides a scheme for synthesizing N-substituted brassilexinderivatives of the present invention.

FIG. 16 provides a scheme for synthesizing cyclopropyl tryptophanderivatives of the present invention.

FIG. 17 provides a scheme for synthesizing aziridinyl tryptophanderivatives of the present invention.

FIG. 18 provides a scheme for synthesizing tetheredcompetitive/noncompetitive derivatives of the present invention.

FIGS. 19A and 19B are graphs showing the results of an enzyme assay forIDO inhibitors. Global nonlinear regression analysis of enzyme kineticdata was obtained for human IDO in the presence of increasingconcentrations of A) 1MT and B) MTH-Trp. Data were plotted and analyzedusing the Prism4 software package (GraphPad). Best fit values of Ki for1MT=34.6 μM and for MTH-Trp=11.4 μM

FIG. 20 is a graph showing the result of a cell-based IDO inhibitorassay of 2 log dose escalation studies for 1MT against IDO, 1MT againstTDO, and MTH-Trp against IDO. Data were plotted using the Prism4 dataanalysis program (GraphPad), and Hillslope and EC50 values weredetermined by nonlinear regression analysis.

FIG. 21 is a schematic diagram of the kynurenine metabolism pathway. IDOis an extrahepatic, interferon-γ inducible oxidoreductase. The productof the IDO reaction, N-formylkynurenine is rapidly hydrolyzed tokynurenine. Kynurenine is distributed between tissue and blood spacesbecause there is little or no activity of the enzymes that furthermetabolize kynurenine in tissues. The major route of kynurenineclearance is excretion in urine after conversion to xanthurenic acid inthe liver and/or kidneys, although it is also an intermediate in thebiosynthetic pathway that produces nicotinamide adenine dinucleotide(NAD) (Takikawa et al. (1986) J. Biol. Chem. 261:3648-3653; Thomas andStocker (1999) Redox. Report 4:199-220).

FIGS. 22A-D are chromatograms showing the results of HPLC analysis ofmouse serum. Serum was prepared by incubating collected blood samples at4° C. overnight and removing the clot. Protein was removed by TCAprecipitation. Samples were resolved on a 250 mm×4.5 mm Luna 5μ C18column (Phenomenex) in isocratic buffer consisting of 20% MeOH, 5%acetonitrile, 10 mM KPO₄ (pH 5.3), 0.15 mM EDTA. Serum samples wereprepared from male FVB strain mice treated as follows: A) Untreated(serum was spiked with 30 μM each of the following control compounds;tryptophan, kynurenine, and 1MT); B) Untreated (55 μM serum tryptophanis detectable); C) LPS challenged for 24 hrs. (induction of kynurenineto 5.6 μM is detectable), D) 1MT pellets implanted subcutaneously for 3days (104 μM 1MT is detectable). Output sensitivity (Y-axis) has beenadjusted at 6.66 and 14.5 minutes to optimize for each anticipated peakheight. Ky=kynurenine, W=tryptophan, 1MT=1-methyl-tryptophan.

FIG. 23 is a graph illustrating the fold change in tumor volume ofMMTVneu mice either mock treated (untreated) or treated with 1MT,L-744,832 (FTI), 1MT and L-744,832, and chemotherapeutic agents with orwithout 1MT. Each data point was determined from an individual mouse andthe bars indicate the mean of the data points as listed at the bottom ofthe graph.

FIG. 24 is a graph of the results from an in vitro biochemical assay forscreening of IDO inhibitor candidates. Data is provided relative to theamount of kynurenine produced in the absence of inhibitor.

FIGS. 25A and 25B are graphs of the results from the cell-based assayfor screening of IDO inhibitor candidates. In FIG. 25A, data is providedrelative to the amount of kynurenine produced in the absence ofinhibitor. In FIG. 25B, the data is presented in terms of fluorescence,which is indicative of kynurenine production (i.e., IDO activity). Cellswere either transfected with an empty expression vector (vector) or anexpression vector containing the cDNA of IDO.

FIG. 26 provides graphs of the thiohydantoin derivatives of indoleaminein the cell-based assay for screening of IDO inhibitor candidates. Thecells were transfected with empty expression vectors (vector) or withexpression vectors which contain IDO or TDO. For comparison, cellstransfected with the IDO expression vector were also assayed in thepresence of 1MT.

FIGS. 27A-27C provide a chart of certain IDO inhibitors, theirstructures, and their ability to inhibit IDO and TDO activity at aconcentration of 250 μM in a cell-based assay.

FIGS. 28A-28B provide graphs demonstrating the toxicity of certain IDOinhibitors of neoplastically transformed breast (top panel) or prostate(bottom panel) cancer cells. Cells were either untreated (Untx) ortreated with 100 μM of inhibitor.

FIG. 29 is a graph illustrating the fold change in tumor volume ofMMTVneu mice either mock treated (untreated) or treated with 1MT,paclitaxel (Taxol®), 1MT and paclitaxel (Taxol®), cisplatin, or 1MT andcisplatin. Each data point was determined from an individual mouse andthe bars indicate the mean of the data points as listed at the bottom ofthe graph.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, a group of novel compoundsexhibiting IDO inhibitory activity are provided. Also encompassed withinthe invention are pharmaceutical compositions comprising such compoundsand methods of use thereof for inhibiting tumor growth.

In yet another embodiment of the present invention, a combinationtreatment protocol comprising administration of an IDO inhibitor with asignal transduction inhibitor (STI) is provided, which is effective forsuppressing tumor growth.

In accordance with yet another aspect of the present invention, anothercombination treatment protocol comprising administration of an IDOinhibitor with a chemotherapeutic agent is provided, which is effectivefor suppressing tumor growth.

In still another embodiment of the present invention, a combinationtreatment protocol is provided for the treatment of a chronic viralinfection, comprising the administration of an IDO inhibitor and achemotherapeutic agent.

I. Definitions

The term “IDO inhibitor” refers to an agent capable of inhibiting theactivity of indoleamine 2,3-dioxygenase (IDO) and thereby reversingIDO-mediated immunosuppression. An IDO inhibitor may be a competitive,noncompetitive, or irreversible IDO inhibitor. “A competitive IDOinhibitor” is a compound that reversibly inhibits IDO enzyme activity atthe catalytic site (for example, without limitation,1-methyl-tryptophan); “a noncompetitive IDO Inhibitor” is a compoundthat reversibly inhibits IDO enzyme activity at a non-catalytic site(for example, without limitation, norharman); and “an irreversible IDOinhibitor” is a compound that irreversibly destroys IDO enzyme activityby forming a covalent bond with the enzyme (for example, withoutlimitation, cyclopropyl/aziridinyl tryptophan derivatives).

IDO inhibitors of the instant invention may include, without limitation,i) previously established (known) IDO inhibitors, such as, but notlimited to: 1-methyl-DL-tryptophan (1MT; Sigma-Aldrich; St. Louis, Mo.),β-(3-benzofuranyl)-DL-alanine (Sigma-Aldrich),beta-(3-benzo(b)thienyl)-DL-alanine (Sigma-Aldrich),6-nitro-L-tryptophan (Sigma-Aldrich), indole 3-carbinol (LKTLaboratories; St. Paul, Minn.), 3,3′-diindolylmethane (LKTLaboratories), epigallocatechin gallate (LKT Laboratories),5-Br-4-Cl-indoxyl 1,3-diacetate (Sigma-Aldrich), 9-vinylcarbazole(Sigma-Aldrich), acemetacin (Sigma-Aldrich), 5-bromo-DL-tryptophan(Sigma-Aldrich), and 5-bromoindoxyl diacetate (Sigma-Aldrich); and ii)the novel IDO inhibitors of the instant invention. In a preferredembodiment of the invention, the IDO inhibitors include the novel IDOinhibitors of the present invention.

A “signal transduction inhibitor” is an agent that selectively inhibitsone or more vital steps in signaling pathways, in the normal function ofcancer cells, thereby leading to apoptosis.

Signal transduction inhibitors (STIs) of the instant invention include,but are not limited to, (i) bcr/abl kinase inhibitors such as, forexample, STI 571 (Gleevec); (ii) epidermal growth factor (EGF) receptorinhibitors such as, for example, kinase inhibitors (Iressa, SSI-774) andantibodies (Imclone: C225 [Goldstein et al. (1995), Clin Cancer Res.1:1311-1318], and Abgenix: ABX-EGF); (iii) her-2/neu receptor inhibitorssuch as, for example, Herceptin™ (trastuzumab), and farnesyl transferaseinhibitors (FTI) such as, for example, L-744,832 (Kohl et al. (1995),Nat Med. 1(8):792-797); (iv) inhibitors of Akt family kinases or the Aktpathway, such as, for example, rapamycin (see, for example, Sekulic etal. (2000) Cancer Res. 60:3504-3513); (v) cell cycle kinase inhibitorssuch as, for example, flavopiridol and UCN-01 (see, for example,Sausville (2003) Curr. Med. Chem. Anti-Canc Agents 3:47-56); and (vi)phosphatidyl inositol kinase inhibitors such as, for example, LY294002(see, for example, Vlahos et al. (1994) J. Biol. Chem. 269:5241-5248).

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount sufficient to modulate tumor growth ormetastasis in an animal, especially a human, including withoutlimitation decreasing tumor growth or size or preventing formation oftumor growth in an animal lacking any tumor formation prior toadministration, i.e., prophylactic administration.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, excipient,auxilliary agent or vehicle with which an active agent of the presentinvention is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

“Concurrently” means (1) simultaneously in time, or (2) at differenttimes during the course of a common treatment schedule.

“Sequentially” refers to the administration of one component of themethod followed by administration of the other component. Afteradministration of one component, the next component can be administeredsubstantially immediately after the first component, or the nextcomponent can be administered after an effective time period after thefirst component; the effective time period is the amount of time givenfor realization of maximum benefit from the administration of the firstcomponent.

“Hydrocarbyl” refers' to an unsubstituted or substituted hydrocarbonmoiety containing from about 1 to about 10 carbon atoms or from about 1to about 25 carbon atoms, which may be a straight, branched, or cyclichydrocarbon group. When substituted, hydrocarbyl groups may besubstituted at any available point of attachment. Exemplaryunsubstituted groups include alkyls such as methyl, ethyl, propyl,isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl,dodecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl,tetracosyl, pentacosyl, and the like; aryls such as phenyl, tolyl,xylyl, napthyl, biphenyl, tetraphenyl, and the like; aralkyls such asbenzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, napthoctyl, and thelike; and cycloalkyls such as cyclopropyl, cyclobutyl, cyclohexyl,cycloheptyl, cyclooctyl, and the like. Exemplary substituents mayinclude but are not limited to one or more of the following groups: halo(such as F, Cl, Br, I), haloalkyl (such as CCl₃ or CF₃), alkoxy,alkylthio, hydroxy, carboxy (—COOH), carbonyl (—C(═O)), epoxy,alkyloxycarbonyl (—C(═O)—OR), alkylcarbonyloxy (—OC(═O)—R), amino(—NH₂), carbamoyl (NH₂C(═O)— or NHRC(═O)—), urea (—NHCONH₂), alkylurea(—NHCONHR) or thiol (—SH), wherein R in the aforementioned substituentsrepresents a hydrocarbyl moiety. Hydrocarbyl groups (moieties) asdefined herein may also comprise one or more carbon to carbon doublebonds or one or more carbon to carbon triple bonds (i.e., thehydrocarbyl groups may be unsaturated). Exemplary unsaturatedhydrocarbyl groups include alkenyls such as allyl and vinyl. Hydrocarbylgroups may also be interrupted with at least one oxygen, nitrogen, orsulfur atom.

The terms “halogen,” “halo,” and “halide” refer to chlorine, bromine,fluorine or iodine.

“Lower alkyl” refers to an alkyl having about 1 to 4 carbon atoms, suchas methyl, ethyl, propyl, buytl, and isopropyl. In a certain embodiment,the lower alkyl is methyl.

II. Novel Compounds Exhibiting IDO Inhibitory Activity and Methods ofUse

In accordance with the instant invention, novel compounds of thefollowing formulas, which are capable of inhibiting IDO activity andthereby suppressing tumor growth, are provided:

formula (I):

wherein R₁ is H or lower alkyl; R₂ is H; R₃ is selected from the groupconsisting of: (a)

wherein R_(A) and R_(B) are independently selected from the group of Hand hydrocarbyl; (b)

wherein R_(C) is selected from the group of H and hydrocarbyl;

wherein n is a whole number from 1 to 10 and R_(D) is a carbolinesubstituent of the formula:

wherein R_(A) and R_(B) are independently selected from the group of Hand hydrocarbyl; or R₂ and R₃ are joined together and represent part ofa ring which is fused to the pyrrole moiety of formula (I) and which isselected from the group of:

wherein R_(E) is a hydrocarbyl or alkyl-Q, Q representing a substituentof the formula:

the compound of formula (I) being a β-carboline derivative when R₂ andR₃ joined together represent (i), a brassilexin derivative when R₂ andR₃ joined together represent (ii), and an N-substituted brassilexinderivative when R₂ and R₃ joined together represent (iii); X, Y, and Zmay be the same or different and are selected from the group consistingof H, halogen, NO₂, and hydrocarbyl; and when R₂ and R₃ are joinedtogether and represent part of a ring system, Y may also beisothiocyanate; with the proviso that formula (I) does not include acompound selected from the group of: 3-(N-methyl-thiohydantoin)-indole,3-(N-phenyl-thiohydantoin)-indole, 3-(N-allyl-thiohydantoin)-indole,5-methyl-brassinin, brassinin, brassilexin, β-carboline,3-butyl-β-carboline, 6-fluoro-3-carbomethoxy-β-carboline,6-isothiocyanate-3-carbomethoxy-β-carboline, 3-propoxy-β-carboline,3-carboxy-β-carboline, 3-carbopropoxy-β-carboline, and3-carbo-tert-butoxy-β-carboline; and formula (II):

wherein X, Y, and Z may be the same or different and are selected fromthe group consisting of H, halogen, NO₂, and hydrocarbyl; and with theproviso that formula (II) does not include 3-amino-2-naphthoic acid.

In one embodiment of the invention, the novel compounds of formula (I)may be further characterized by the following additional provisos: 1) ifR₃ is substituent (a) and if R_(A) is other than H, then either R_(B) ishydrocarbyl, or at least one of X, Y, or Z is other than H, NO₂, COH,CH₂OH, or CH₃; 2) if R₃ is substituent (a) and if R_(B) is H, theneither R_(A) is H, or at least one of X, Y, or Z is other than H, NO₂,COH, CH₂OH, or CH₃; 3) if R₃ is substituent (a) and if X, Y, and Z areall selected from the group consisting of H, NO₂, COH, CH₂OH, or CH₃,then either R_(A) is H, or R_(B) is hydrocarbyl.

In yet another embodiment of the invention, the novel compounds offormula (I) may be further characterized by the additional proviso thatwhen R₃ is (f), Y is a halogen.

In a particular embodiment of the invention, when R₃ is (f), X and Z areH, Y is Br, R₁ is —CH₃, R₂ is H, R_(A) is —CH₃ or —CH₂—CH═CH₂, and R_(B)is H.

The compounds of formulas (I) wherein R₃ is selected from the groupconsisting of (a), (b), and (f) and formula (II) are believed tocompetitively inhibit IDO activity, while the compounds of formulas (I)wherein R₃ is selected from the group consisting of (c), (d), and (e) orR₂ and R₃ are joined to form one of the group consisting of (i), (ii),and (iii) likely inhibit IDO activity noncompetitively.

The present invention also provides pharmaceutical compositionscomprising at least one of the IDO inhibitors having a formula selectedfrom formulas (I) and (II) in a pharmaceutically acceptable carrier.Such a pharmaceutical composition may be administered, in atherapeutically effective amount, to a patient in need thereof for thetreatment of cancer.

Moreover, the present invention provides a method for the treatment ofcancer by administering to a patient, in need thereof, a therapeuticallyeffective amount of at least one IDO inhibitor having the formulaselected from formulas (I) and (II).

Cancers that may be treated using the present protocol include, but arenot limited to: cancers of the prostate, colorectum, pancreas, cervix,stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck,skin (including melanoma and basal carcinoma), mesothelial lining, whiteblood cell (including lymphoma and leukemia) esophagus, breast, muscle,connective tissue, lung (including small-cell lung carcinoma andnon-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone;glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma,sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, andtesticular seminoma.

III. Combinatorial Therapies for the Treatment of Cancer

The present invention also provides methods for tumor suppression. Inaccordance with the present invention, it has been discovered that thecombination of an IDO inhibitor with a signal transduction inhibitor(STI) act synergistically to suppress tumor growth. Accordingly, thepresent invention provides a pharmaceutical composition for thetreatment of cancer in a patient comprising at least one IDO inhibitorand at least one STI in a pharmaceutically acceptable carrier. Alsoprovided is a method for treating cancer in a patient by administeringan effective amount of at least one IDO inhibitor in combination with atleast one STI. Suitable IDO inhibitors include any compound whichexhibits IDO inhibitory activity including compounds having a formulaselected from formulas (I) and (II). Suitable STIs, as notedhereinabove, include, but are not limited to: (i) bcr/abl kinaseinhibitors such as, for example, STI 571 (Gleevec); (ii) epidermalgrowth factor (EGF) receptor inhibitors such as, for example, kinaseinhibitors (Iressa, SSI-774) and antibodies (Imclone: C225 [Goldstein etal. (1995), Clin Cancer Res. 1:1311-1318], and Abgenix: ABX-EGF); (iii)her-2/neu receptor inhibitors such as, for example, Herceptin™(trastuzumab) and farnesyl transferase inhibitors (FTI) such as, forexample, L-744,832 (Kohl et al. (1995), Nat. Med. 1(8):792-797); (iv)inhibitors of Akt family kinases or the Akt pathway, such as, forexample, rapamycin (see, for example, Sekulic et al. (2000) Cancer Res.60:3504-3513); (v) cell cycle kinase inhibitors such as, for example,flavopiridol and UCN-01 (see, for example, Sausville (2003) Curr. Med.Chem. Anti-Canc Agents 3:47-56); and (vi) phosphatidyl inositol kinaseinhibitors such as, for example, LY294002 (see, for example, Vlahos etal. (1994) J. Biol. Chem. 269:5241-5248).

In a specific embodiment of the present invention, the at least one IDOinhibitor and at least one STI may be administered to the patientconcurrently or sequentially. In other words, the at least one IDOinhibitor may be administered first, the at least one STI may beadministered first, or the at least one IDO inhibitor and the at leastone STI may be administered at the same time. Additionally, when morethan one IDO inhibitor and/or STI is used, the compounds may beadministered in any order.

Cancers that may be treated using the present combinatorial protocolinclude, but are not limited to: cancers of the prostate, colorectum,pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary,testis, head, neck, skin (including melanoma and basal carcinoma),mesothelial lining, white blood cell (including lymphoma and leukemia)esophagus, breast, muscle, connective tissue, lung (including small-celllung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma,gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellularcarcinoma, and testicular seminoma.

In addition to IDO, other molecules are known to be involved inimmunomodulation. These other molecules may also be potential targetsfor suppressing tumor growth in cancer patients. Accordingly, thepresent invention also provides combinatorial methods of treating cancerpatients by the administration of at least one immunomodulator otherthan an IDO inhibitor in conjunction with at least one chemotherapeuticagent. Suitable immunomodulators that may be used in the presentinvention include, without limitation: costimulatory molecules, such as,CD40L, B7, and B7RP1; activating monoclonal antibodies (mAbs) tocostimulatory receptors, such as, ant-CD40, anti-CD38, anti-ICOS, and4-IBB ligand; dendritic cell antigen loading (in vitro or in vivo);dendritic cell cancer vaccine; cytokines/chemokines, such as, IL1, IL2,IL12, IL18, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC,IFNa/b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacteriallipopolysaccharides (LPS); and immune-stimulatory oligonucleotides(e.g., poly CpG DNA (see, for example, Verthelyi and Zeuner (2003) Tr.Immunol. 24:519-522)). Suitable chemotherapeutic agents include, but arenot limited to, cytotoxic chemotherapeutic agents and signaltransduction inhibitors (STIs).

In accordance with the present invention, it has also been discoveredthat the combination of an IDO inhibitor with a chemotherapeutic agentact synergistically to suppress tumor growth. Accordingly, the presentinvention provides a pharmaceutical composition for the treatment ofcancer in a patient comprising at least one IDO inhibitor and at leastone chemotherapeutic agent in a pharmaceutically acceptable carrier.Also provided is a method for treating cancer in a patient byadministering an effective amount of at least one IDO inhibitor incombination with at least one chemotherapeutic agent. Suitable IDOinhibitors include any compound which exhibits IDO inhibitory activityincluding compounds having a formula selected from formulas (I) and(II). Suitable chemotherapeutic agents, as noted hereinabove, include,but are not limited to: paclitaxel (Taxol®), cisplatin, docetaxol,carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide,CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine,adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, andepothilone derivatives.

In a specific embodiment of the present invention, the at least one IDOinhibitor and at least one chemotherapeutic agent may be administered tothe patient concurrently or sequentially. In other words, the at leastone IDO inhibitor may be administered first, the at least onechemotherapeutic agent may be administered first, or the at least oneIDO inhibitor and the at least one chemotherapeutic agent may beadministered at the same time. Additionally, when more than one IDOinhibitor and/or chemotherapeutic agent is used, the compounds may beadministered in any order.

Cancers that may be treated using the present combinatorial protocolinclude, but are not limited to: cancers of the prostate, colorectum,pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary,testis, head, neck, skin (including melanoma and basal carcinoma),mesothelial lining, white blood cell (including lymphoma and leukemia)esophagus, breast, muscle, connective tissue, lung (including small-celllung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma,gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellularcarcinoma, and testicular seminoma.

IV. Combinatorial Therapy for the Treatment of Chronic Viral Infections

The present invention further provides a pharmaceutical composition forthe treatment of a chronic viral infection in a patient comprising atleast one IDO inhibitor, at least one cancer therapeutic drug in apharmaceutically acceptable carrier, and, optionally, at least oneantiviral agent. Also provided is a method for treating a chronic viralinfection in a patient by administering an effective amount thecomposition just described.

Suitable IDO inhibitors include any compound which exhibits IDOinhibitory activity including compounds having a formula selected fromformulas (I) and (II).

Suitable chemotherapeutic agents are any compounds that exhibitanticancer activity including, but not limited to: alkylating agents(e.g., nitrogen mustards such as chlorambucil, cyclophosphamide,isofamide, mechlorethamine, melphalan, and uracil mustard; aziridinessuch as thiotepa; methanesulphonate esters such as busulfan; nitrosoureas such as carmustine, lomustine, and streptozocin; platinumcomplexes such as cisplatin and carboplatin; bioreductive alkylatorssuch as mitomycin, procarbazine, dacarbazine and altretamine); DNAstrand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors(e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone,doxorubicin, etoposide, and teniposide); DNA minor groove binding agents(e.g., plicamydin); antimetabolites (e.g., folate antagonists such asmethotrexate and trimetrexate; pyrimidine antagonists such asfluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, andfloxuridine; purine antagonists such as mercaptopurine, 6-thioguanine,fludarabine, pentostatin; asparginase; and ribonucleotide reductaseinhibitors such as hydroxyurea); tubulin interactive agents (e.g.,vincristine, vinblastine, and paclitaxel (Taxol)); hormonal agents(e.g., estrogens; conjugated estrogens; ethinyl estradiol;diethylstilbesterol; chlortrianisen; idenestrol; progestins such ashydroxyprogesterone caproate, medroxyprogesterone, and megestrol; andandrogens such as testosterone, testosterone propionate,fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g.,prednisone, dexamethasone, methylprednisolone, and prednisolone);leutinizing hormone releasing agents or gonadotropin-releasing hormoneantagonists (e.g., leuprolide acetate and goserelin acetate); andantihormonal antigens (e.g., tamoxifen, antiandrogen agents such asflutamide; and antiadrenal agents such as mitotane andaminoglutethimide). Preferably, the chemotheraputic agent is selectedfrom the group consisting of: paclitaxel (Taxol®), cisplatin, docetaxol,carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide,CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine,adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, andepothilone derivatives.

Suitable antiviral agents include, without limitation: acyclovir;gangcyclovir; foscarnet; ribavirin; and antiretrovirals such as, forexample, nucleoside analogue reverse transcriptase inhibitors (e.g.,azidothymidine (AZT), ddI, ddC, 3TC, d4T), non-nucleoside reversetranscriptase inhibitors (e.g., efavirenz, nevirapine), nucleotideanalogue reverse transcriptase inhibitors, and protease inhibitors.

In a specific embodiment of the present invention, the at least one IDOinhibitor and at least one chemotherapeutic agent may be administered tothe patient concurrently or sequentially. In other words, the at leastone IDO inhibitor may be administered first, the at least onechemotherapeutic agent may be administered first, or the at least oneIDO inhibitor and the at least one STI may be administered at the sametime. Additionally, when more than one IDO inhibitor and/orchemotherapeutic agent is used, the compounds may be administered in anyorder. Similarly, the antiviral agent may also be administered at anypoint.

The compounds of this combination treatment may also be administered forlocalized infections. Specifically, the at least one IDO inhibitor, atleast one chemotherapeutic agent, and, optionally, at least oneantiviral agent may be administered to treat skin infections such asshingles and warts. The compounds may be administered in anypharmaceutically acceptable topical carrier including, withoutlimitation: gels, creams, lotions, ointments, powders, aerosols andother conventional forms for applying medication to the skin.

Chronic viral infections that may be treated using the presentcombinatorial treatment include, but are not limited to, diseases causedby: hepatitis C virus (HCV), human papilloma virus (HPV),cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus(EBV), varicella zoster virus, coxsackie virus, human immunodeficiencyvirus (HIV).

Notably, parasitic infections (e.g. malaria) may also be treated by theabove methods wherein compounds known to treat the parasitic conditionsare optionally added in place of the antiviral agents.

V. Administration of Pharmaceutical Compositions and Compounds

The pharmaceutical compositions of the present invention can beadministered by any suitable route, for example, by injection, by oral,pulmonary, nasal or other modes of administration. In general,pharmaceutical compositions of the present invention, comprise, amongother things, pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionscan include diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; and additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol). The compositions can be incorporated into particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc., or into liposomes. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of components of a pharmaceutical compositionof the present invention. See, e.g., Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712 which are herein incorporated by reference. The pharmaceuticalcomposition of the present invention can be prepared, for example, inliquid form, or can be in dried powder form (e.g., lyophilized).

In yet another embodiment, the pharmaceutical compositions of thepresent invention can be delivered in a controlled release system, suchas using an intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In aparticular embodiment, a pump may be used (see Langer, supra; Sefton,CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery(1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). Inanother embodiment, polymeric materials may be employed (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Press:Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley: New York (1984);Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. (1983) 23:61;see also Levy et al., Science (1985) 228:190; During et al., Ann.Neurol. (1989) 25:351; Howard et al., J. Neurosurg. (1989) 71:105). Inyet another embodiment, a controlled release system can be placed inproximity of the target tissues of the animal, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, (1984) vol. 2, pp. 115-138).In particular, a controlled release device can be introduced into ananimal in proximity to the site of inappropriate immune activation or atumor. Other controlled release systems are discussed in the review byLanger (Science (1990) 249:1527-1533).

The following examples are provided to illustrate various embodiments ofthe present invention. These examples are not intended to limit theinvention in any way.

Example 1 Synthesis of Thiohydantoin Derivatives

Overview:

Thiohydantoin derivatives, or compounds of formula (I) wherein R₃ isgroup (a), were identified by screening commercially availabletryptophan derivatives. N-Methyl (R₁=H; R_(A)=CH₃; R_(B)=H; X, Y, Z=H),N-allyl (R₁=H; R_(A)=—CH₂CH═CH₂; R_(B)=H; X, Y, Z=H) and N-phenyl (R₁=H;R_(A)=Ph; R_(B)=H; X, Y, Z=H) thiohydantoin derivatives demonstratedgreater than 50% inhibition and significant selectivity over thestructurally distinct enzyme tryptophan 2,3-dioxygenase (TDO2). Tofurther improve inhibitor potency, the focus of the present inventioncan be on substitution of the indole ring, a procedure which hasenhanced potency of tryptophan derivatives in the past. In particular,literature reports indicate that N-1, C-5 and C-6 substitution of theindole ring will afford more potent inhibitors (Cady and Sono (1991)Arch. Biochem. Biophys., 291:326-33; Munn, D. H., et al. (1998) Science,281:1191-3; Peterson, A. C., et al. (1994) Med. Chem. Res., 3:531-544;Southan, M. D., et al. (1996) Med. Chem. Res., 6: 343-352).Additionally, while C-4 halide derivatives have not been reported in theliterature (Southan et al.), the instant invention has identified5-bromo-4-chloroindoxyl 1,3-diacetate as a potent inhibitor (greaterthan 50% inhibition of IDO at 250 μM).

Synthesis:

Thiohydantoin derivatives can be synthesized via an Erlenmeyer-Plochlazlactone-type condensation reaction between an indole-3-carboxaldehydeand thiohydantoin 1 (FIG. 1; Djura and Faulkner (1980) J. Org. Chem.,45:735-7; Guella, G., et al. (1988) Hely. Chim. Acta, 71:773-82; Guella,G., et al. (1989) Hely. Chim. Acta, 72:1444-50; Crawford and Little(1959) J. Chem. Soc., 729-731; Julian and Sturgis (1935) J. Am. Chem.Soc., 1126-28). A mixture of conjugated alkene stereoisomers 3 willlikely result and these products can be tested as planar analogues ofthe thiohydantoins. Reduction of the conjugated alkene in 3 can beperformed by nucleophilic reductants (e.g. NaBH₄, Li(s-Bu)₃BH, and[(Ph₃P)CuH]₆) as the presence of sulfur will prohibit more commonly usedcatalytic hydrogenation methods.

The thiohydantoins can be synthesized by the reaction of, for example,glycine methyl ester or glycine ethyl ester with alkyl isothiocyanates(FIG. 1; Sim and Ganesan (1997) J. Org. Chem., 62:3230-35). Notably,this method of thiohydantoin synthesis allows for a variety of alkylgroups to be incorporated at the N-1 nitrogen atom via reductiveamination (Sim and Ganesan (1997) J. Org. Chem., 62:3230-35) and N-3nitrogen atom depending on the choice of isothiocyanate reagent.

Based on previous IDO inhibitor studies, a variety of halo-substitutedindole-3-carboxaldehydes can be used in the condensation reactions withthiohydantoins. 5-Bromo- and 5-chloroindole-3-carboxaldehyde arecommercially available. Substitution in the C-4 position of theindole-3-carboxaldehyde can be accomplished by thallation-halogenationof indole-3-carboxaldehydes 5 (FIG. 2; Somei, M., et al. (1984)Heterocycles, 22(4):797-801; Ohta, T., et al. (1987) Hetereocycles26:2817-22; Somei, M., et al. (1985) Chem. Pharm. Bull. 33:3696-708).N-methylation of the indole-3-carboxaldehydes 6 can be performed byreacting with a dimethyl carbonate (Jiang, X., et al. (2001) Org. Proc.Res. Dev., 5:604-8).

Generation of 6-halo-indole-3-carboxaldehydes can proceed via6-aminoindole, which is readily synthesized from commercially available2,4-dinitrotoluene via a Leimgruber-Batcho indole synthesis (FIG. 3;Batcho and Leimgruber (1990) Org. Syn. Coll., 3:34). The 6-amino groupcan be protected as an acetamide. Vilsmeier formylation of the C-3position can provide the 6-substituted indole-3-carboxaldehyde 10(Smith, G. F. (1954) J. Chem. Soc., 3842-6; James and Snyder (1963) Org.Syn. Coll., 4:539). The presence of an electron-releasing acetamidegroup can enhance the efficiency of the Vilsmeter formylation.Methylation of the indole nitrogen can proceed as described previously.The 6-amino group can be deprotected under acid reflux conditions andthen the amine 12 can be transformed into a halogen via Sandmeyer orSchiemann reaction (Somei, M., et al. (1985) Chem. Pharm. Bull.,33:3696-708; Somei and Tsuchiya (1981) Chem. Pharm. Bull., 29:3145-57;Moriya, T., et al. (1975) Bull. Chem. Soc. Jpn., 48:2217-8) or oxidizedto a nitro group with a peroxyacid (Pagano and Emmons, Org. Syn. Coll.,5:367). Problems with side reactions involving the 3-carboxaldehydeduring the Sandmeyer, Schiemann or amine oxidation reactions can beovercome by altering the sequence to introduce the 3-carboxaldehyde andmethylate at the N-1 position later.

Derivatives with substitution at both the 5 and 6 positions can besynthesized via electrophilic aromatic substitution of 14 (FIG. 4).Nitration of 1-methyl-indole-3-carboxaldehyde has reportedly afforded a93% yield of a mixture of the 5-nitro and 6-nitro products (Da Settimoand Saettone (1965) Tetrahedron 21:1923-9). Use of the 6-acetamidoderivative prevents nitration at the 6 position and further activatesthe 5 position to eletrophilic aromatic substitution. In addition tonitration, bromination and chlorination may also be employed and therebyallow a variety of 5,6-disubstituted indole-3-carboxaldehydes to besynthesized. In addition, thallation-halogenation (Somei, M., at al.(1984) Heterocycles, 22(4):797-801; Ohta, T., et al. (1987)Heterocycles, 26:2817-22; Somei, M., et al. (1985) Chem. Pharm. Bull.,33:3696-708) of the 5,6-disubstituted indole-3-carboxaldehydes 15permits functionalization of the C-4 position. Furthermore, followingthe methods elaborated in Scheme 3 (FIG. 3), the C-4 acetamide group canbe modified. When combined, these procedures allow complexpolysubstituted indole-3-carboxaldehydes to be obtained in a highlyefficient manner.

Notably, IDO demonstrates a preference for L-tryptophan (Peterson, A.C., et al. (1994) Med. Chem. Res., 3:531-544), but will also acceptD-tryptophan as a substrate (Sono, M. (1989) Biochemistry, 28:5400-7).Therefore, an enantiomerically pure product may not be absolutelyrequired.

An alternative approach for the synthesis of thiohydantoin derivativesinvolves the condensation of 2 with the ethyl half ester ofacetamidomalonic acid (FIG. 5; Hengartner, U., et al. (1979) J. Org.Chem., 44:3741-7). Hydrogenation with Wilkinson's catalyst generates 19(Horwell, D. C., et al. (1994) J. Org. Chem., 59:4418-23). Hydrolysis ofthe acetamide and ethyl ester affords the tryptophan analogue 20.Reaction with alkyl isothiocyanate affords the thiohydantoin product(Cejpek, K., et al. (2000) J. Agric. Food. Chem., 48:3560-5; Mizrach andPolonska (1987) Khim. Farm. Zh., 21:322-8), although it may be moreefficient to perform the isothiocyanate reaction on the analogous aminoester (Sim and Ganesan (1997) J. Org. Chem., 62:3230-35).

A potentially important benefit of the alternative path is the abilityto obtain an enantiomeriaclly enriched product. Several asymmetrichydrogenations of related dehydrotryptopha compounds have been reported(Hengartner, U., et al. (1979) J. Org. Chem., 44:3741-7; Knowles, W. S.,et al. (1975) J. Am. Chem. Soc., 97:2567-8). Therefore, substitution ofthe triphenylphosphine ligands of Wilkinson's catalyst with chiralphosphine ligands can result in stereoselective synthesis of the Lisomer of the tryptophan analogue.

Example 2 Synthesis of Brassinin Derivatives

Overview:

Brassinin and related phytoalexins have demonstrated anticancerproperties, reportedly due to preventative effects achieved bycarcinogen detoxification (Park and Pezzuto (2002) Cancer MetathesisRev., 21:231-255; Mehta, R. G., et al. (1995) Carcinogenesis 16:399-404;Mehta, R. G., et al. (1994) Anticancer Research 14:1209-1213).Interestingly, brassilexin, a related phytoalexin, is reportedly anoncompetitive inhibitor of IDO (Peterson, A. C., et al. (1993) Med.Chem. Res., 3:473-482), whereas, preliminary work indicates brassinin isa competitive inhibitor of IDO. To date, there have been no studies ofIDO inhibition with brassinin derivatives (i.e. compounds having theformula of formula (I) wherein R₃ is group (b) or brassilexinderivatives (i.e., compounds having the formula of formula (I) whereinR₃ and R₂ are joined to form (ii) or (iii)). The structural similarityof brassinin to tryptophan and its identification as a competitiveinhibitor suggest derivatization similar to tryptophan-based inhibitorsalready reported.

Synthesis:

Indole-3-carboxaldehydes discussed above (FIGS. 2-4) can be employed togenerate brassinin derivatives, i.e., IDO inhibitors having the formulaof formula (I) where R₃ is group (b). The indole-3-carboxaldehydes 2 canbe transformed into 3-aminomethyl derivatives 22 by reductive aminationwith hydroxylamine (FIG. 6). The 3-aminomethylindole derivatives may beunstable (Kutschy, P., et al. (1991) Synlett, 289-290; Kutschy, P., etal. (1998) Tet. 54:3549-66) and therefore will immediately be treatedwith carbon disulfide and then methyl iodide to generate brassininderivatives with substitution in the benzene ring (Takasugi, M., et al.(1988) Bull. Chem. Soc. Jpn. 61:285; Pedras, M. S. C., et al. (1992)Life Science Advances (Phytochemistry), 11:1; Mehta, R. G., et al.(1995) Carcinogenesis, 16(2):399-404).

Example 3 Synthesis of β-Carboline Derivatives

Overview:

β-Carboline or norharman derivatives, i.e., compounds having thestructure of formula (I) wherein R₃ and R₂ are joined to form (i), havebeen previously reported (Sono and Cady (1989) Biochemistry,28:5392-5399; Peterson, A. C., et al. (1993) Med. Chem. Res., 3:473-482;Eguchi, N., et al. (1984) Arch. Biochem. Biophys., 232(2):602-609).However, analogues of the most active derivative, 3-butyl-β-carboline,have not been reported. Notably, substitution at the C-6 position (—Fand —N═C═S) of the β-carboline core yielded several similarly activederivatives, but analogues that combine the C-3 and C-6 substitutionwere not tested; nor were any other substitutions larger than the C-3butyl of the β-carboline explored (Peterson, A. C., et al. (1993) Med.Chem. Res. 3:473-482). β-carboline derivatives with substitution at theC-5, 7, and 8 position have also not been reported. Of particularinterest is the reported mechanism of action of β-carboline derivativesas non-competitive inhibitors, e.g., competition with oxygen for abinding site on the heme iron of the ferrous enzyme (Sono and Cady(1989) Biochemistry, 28:5392-5399).

Synthetic:

The commercially availability of ethyl β-carboline-3-carboxylate 24makes chemical modifications of this structure attractive for thesynthsis of β-carboline derivatives (FIG. 7). Nitration (Settimj, G., etal. (1988) J. Heterocyclic Chem. 25:1391-7) and iodination (Huth, A., etal. (1987) Tet. 43:1071-4) of the C-6 position of 24 has beenaccomplished in good yield. In addition, regioselective bromination(Ponce and Erra-Balsells (2001) J. Heterocyclic Chem. 38:1087-1095;Kardono, L. B. S., et al. (1991) J. Nat. Prod. 54:1360-7) andchlorination (Ponce, M. A., et al. (2003) J. Heterocyclic Chem.40:419-426) procedures for β-carboline have also been reported.Reduction of the C-6 nitration product has been accomplished (Settimj,G., et al. (1988) J. Heterocyclic Chem. 25:1391-7) and the resultingamine has been used to direct bromination in the C-5 position of theβ-carboline (Huth, A., et al. (1987) Tet. 43:1071-4). These reactionswill facilitate the synthesis of C-5 and C-6 substituted β-carbolinederivatives.

Derivatization of the C-3 position may be performed via the C-3carboxaldehyde (FIG. 8). Chemoselective reduction (or reduction to thealcohol and chemoselective oxidation) of the C-3 ester of 24 with DIBAL,affords the C-3 carboxaldehyde. Addition of organolithium or Grignardreagents to the aldehyde affords alcohol derivatives 29. Dehydration ofthe alcohol affords an alkene 30 that can be selectively reduced toyield the C-3 alkyl group of variable length depending on the choice oforganometallic reagent. The alcohol and alkene intermediates may also betested. Alternatively, Wittig reaction with the carboxaldehyde canproduce the alkene.

An alternative approach to β-carboline derivatives involves cyclizationand oxidation of tryptophan derivatives 32 (FIG. 9; Haffer, G., et al.(1998) Heterocycles 48(5):993-8). The tryptophan derivatives discussedearlier (FIGS. 2-4) are amenable to cyclization to form thepyrido-indole structure. One advantage in using this procedure is theability to create potential β-carboline based IDO inhibitors withfunctionalization in the C-7 position of β-carboline.

Example 4 Synthesis of 3-Amino-2-Naphthoic Acid Derivatives

Overview:

In 1994, Peterson and co-workers reported 3-amino-2-naphthoic acid to bethe most potent IDO inhibitor amongst a selection of tryptophanderivatives and related compounds (Peterson, A. C., et al. (1994) Med.Chem. Res. 3:531-544). Notably, there were no other derivatives of3-amino-2-napthoic acid reported in the article, nor, insofar as isknown, have other tests with this structure of related derivatives beenreported, despite the fact that the compound was more potent than theprototypical IDO inhibitor L-1-methyl-tryptophan (1MT). Since3-amino-naphthoic acid is a competitive inhibitor, derivatives withsubstituents in the C-6, C-7, and C-8 positions of the naphthalene core(analogous to the C-4, C-5, and C-6 positions of the indole ring intryptophan) can provide a superior IDO inhibitor.

Synthesis:

Although there exist many methods to synthesize naphthalene compounds(de Koning, C. B., et al. (2003) Tet. 59:7-36), the synthesis ofasymmetrically substituted naphthalenes with substituents in both ringsremains a challenge. However, substituted 3-amino-2-naphthoic acids canbe generated by first performing a benzannulation of substitutedphthaldehydes (Kienzle, F. (1980) Helv. Chim. Acta 63:2364-9; Singh andKhade (2002) Bioconj. Chem. 13:1286-91). The rapid synthsis of3-amino-7-bromo-2-naphthoic acid 36 demonstrates the efficiency of thismethodology (FIG. 10).

The phthaldehyde 34 can be synthesized from 4-bromo-o-xylene or dimethylphthalate. Ethyl 3-nitropropionate can be synthesized in one step fromsilver nitrite and ethyl 3-bromopropionate (Belikov, V. M. (1956) Izv.Akad. Nauk., S. S. S. R.; Otdel. Khim. Nauk 855-62; Chemical abstract(1957) 51:1837j). Condensation of the two affords a mixture of 6-bromoand 7-bromo 3-nitro-2-naphthoic acids 35. Reduction of the nitro groupand hydrolysis of the ester affords the brominated 3-amino-2-naphthoicacids. The two regioisomers may be separated at some point in thesynthesis, but both compounds can also be tested and the rapid manner inwhich the two are assembled likely outweighs any inconveniences of theregioisomeric mixture.

The synthesis of additional substituted 3-amino-2-naphthoic acids allowsfor regiocontrol through the use of electron donating groups in place ofthe bromine (FIG. 11). The phthaldehydes 37 can be generated from either3,4-dimethylaniline or dimethyl phthalate. Electrophilic aromaticsubstitution of the amino or acetamido phthaldehyde precursors allowsfor the incorporation of groups in the C-6 position of the3-amino-2-naphthoic acid. The benzannulation reaction is regioselectivedue to electron releasing acetamido group (Kienzle, F. (1980) Hely.Chim. Acta 63:2364-9). After benzannulation, the 7-acetamido or aminogroup allows for the efficient substitution at C-8 via electrophilicaromatic substitution reactions. Upon deprotection, the 7-amino groupcan undergo diazotization and Sandmeyer or Schiemann reaction to afforda variety of substituents. Nitro group reduction and ester hydrolysisaffords the 3-amino-2-naphthoic acid derivatives, i.e. compounds offormula (II) for testing.

As an alternative method, methoxy phthaldehydes 43 (Z═H) havedemonstrated considerable control in condensation reactions with3-nitropropionates (FIG. 12; Kienzle, F. (1980) Helv. Chim. Acta63:2364-9). The methoxy group directs electrophilic aromaticsubstitution reactions to ortho positions, however to transform themethoxy group into an amine and subsequently to a halogen, one canemploy the Buchwald-Hartwig amination methodology (Wolfe, J. P., et al.(2000) J. Org. Chem. 65:1158-74; Wolfe and Buchwald (1997) J. Org. Chem.62:1264-7; Louie, J., et al. (1997) J. Org. Chem. 62:1268-73; Hartwig,J. F. (1998) Angew. Chem. Int. Ed. Engl. 37:2046-67). Conversion of themethoxy to triflate 45 allows for amination, but carboxylic esters arenot compatible with this process due to the strongly basic conditions.Consequently, 3-nitro-propanenitrile, which can be synthesized similarlyto ethyl 3-nitropropionate but with 3-bromopropionitrile as startingmaterial, can be used in the condensation and the cyano group can behydrolyzed at the end of the synthsis.

Example 5 Synthesis of Brassilexin Derivatives

Brassilexin derivatives, compounds of formula (I), wherein R₃ and R₂ arejoined to form (ii) or (iii) can be synthesized as shown in FIG. 13(see, for example, Devys and Barbier (1993) Org. Prep. Proc. Int.25:344-346).

The synthesis scheme for substituted brasilexin derivatives is shown inFIGS. 14 and 15 (see, for example, Yeung et al. (2002) Tet. Lett.43:5793-5795).

Example 6 Synthesis of Cyclopropyl/Aziridinyl Tryptophan Derivatives

The schemes for synthesizing cyclopropyl/aziridinyl tryptophanderivatives, compounds of formula (I) where R₃ is (c) or (d), are shownin FIGS. 16 and 17 (see, for example, Donati et al. (1996) Tetrahedron52:9901-9908; Ishikawa et al. (2001) J. Am. Chem. Soc. 123:7705-7706).

Example 7 Synthesis of Tethered Competitive/Noncompetitive Derivatives

FIG. 18 shows the scheme for the synthesis of tetheredcompetitive/noncompetitive derivatives, i.e. compounds of formula (I)where R₃ is (e).

Example 8 Synthesis of Didehyro Derivatives

FIG. 1 shows a scheme for the synthesis of didehydro derivatives(compound 3 in FIG. 1), i.e. compounds of formula (I) where R₃ is (f).Example 1 hereinabove provides a more detailed description of thesecompounds. Notably, both E and Z diastereomers are included in theinstant invention.

Example 9 Evaluation of Novel IDO Inhibitors

1. Biochemical Evaluation of Novel IDO Inhibitors:

Overview:

The biochemistry of IDO is well established, the enzyme having firstbeen isolated in 1963 (Higuchi, K., et al. (1963) Federation Proc.22:243 (abstr.); Shimizu, T., et al. (1978) J. Biol. Chem. 253:4700-6).IDO is a monomeric, haem-containing oxidoreductase with a molecularweight of approximately 41 kDa. To maintain the active ferrous formduring in vitro catalysis, the enzyme requires methylene blue incombination with either superoxide or a reductant such as ascorbic acid.In vivo, it is suggested that a flavin or tetrahydrobiopterin mayfulfill the role of the methylene blue dye and that there is likely tobe a specific site for noncompetitive IDO inhibitors. Active enzyme canbe produced by expressing the cloned, His-tagged version of themammalian gene in bacteria (Littlejohn, T. K., et al. (2000) Prot. Exp.Purif. 19:22-29). This provides a convenient source of enzyme forbiochemical analysis. A conventional biochemical assay for IDO activitybased on spectaphotometric measurement of the production of kynurenine(the hydrolysis product of N-formyl-kynurenine) from tryptophan(Daubener, W., et al. (1994) J. Immunol. Methods 168:39-47) is used asthe read-out for both the enzymatic and cell-based assays. The enzymaticassay provides a facile, high-throughput screen for identifyingcompounds with IDO inhibitory activity. This assay is also used todetermine Ki values for specific compounds, which is important for thedevelopment of SAR (structure activity relationship) around thedifferent compound series. The cell-based assay both confirms the IDOinhibitory activity of identified compounds, and addresses the initialissue of bioavailability—the ability of compounds to inhibitintracellular IDO. Specificity for IDO inhibition is examined in thecell-based assay by comparing against the other known tryptophancatabolizing enzyme tryptophan dioxygenase (TDO, also referred to in theliterature as TDO2).

Methods:

cDNA clones for both human and mouse IDO have been isolated and verifiedby sequencing. To prepare enzyme for biochemical studies, C-terminalHis-tagged IDO protein can be produced in E. coli using theIPTG-inducible pET5a vector system and isolated over a nickel column.The yield of the partially purified protein can be verified by gelelectrophoresis and the concentration estimated by comparison to proteinstandards. To assay IDO enzymatic activity, a 96-well platespectraphotometric assay for kynurenine production can be run followingpublished procedures (Littlejohn, T. K., et al. (2000) Prot. Exp. Purif.19:22-29; Takikawa, O., et al. (1988) J. Biol. Chem. 263:2041-8). Toscreen for evidence of IDO inhibitory activity, compounds can beevaluated at a single concentration of, for example, 200 μM against 50ng of IDO enzyme in 100 μl reaction volumes with tryptophan added atincreasing concentrations at, for example, 0, 2, 20, and 200 μM.Kynurenine production can be measured at 1 hour. More extensive enzymekinetic data can be collected for selected compounds of interest. Bestfit Ki value determinations for 1MT (Ki=34.6 μM) and for MTH-Trp(Ki=11.4 μM) are shown in FIG. 19. These data indicate that thethiohydantoin form directly inhibits IDO enzyme activity with about3-fold greater potency than is achieved with 1MT.

The following procedure is an example of a cell-based assay. COS-1 cellsare transiently transfected with a CMV promoter-driven plasmidexpressing IDO cDNA using Lipofectamine 2000 (Invitrogen) as recommendedby the manufacturer. A companion set of cells is transiently transfectedwith TDO-expressing plasmid. 48 hours post-transfection, the cells areapportioned into a 96-well format at 6×10⁴ cells per well. The followingday the wells are washed and new media (phenol red free) containing 20μg/ml tryptophan is added together with inhibitor. The reaction isstopped at 5 hours and the supernatant removed andspectraphotometrically assayed for kynurenine as described for theenzyme assay (Littlejohn, T. K., et al. (2000) Prot. Exp. Purif.19:22-29; Takikawa, O., et al. (1988) J. Biol. Chem. 263:2041-8). Toobtain initial confirmation of IDO activity, compounds can be evaluatedat a single concentration of, for example, 100 μM. More extensive doseescalation profiles can be collected for select compounds. EC50 valuedeterminations for 1MT (EC50=267 μM) and for MTH-Trp (EC50=12.9 μM) areshown in FIG. 20. These data indicate that MTH-Trp is substantially morepotent against intracellular IDO (˜20-fold) than is 1MT.

2. Pharmacodynamic/Pharmacokinetic Evaluation of Novel IDO Inhibitors:

Overview:

Intraperitoneal administration of bacterial lipopolysaccharide (LPS)induces IDO activity in a variety of tissues resulting in the productionof kynurenine and its release into the bloodstream (FIG. 21). Peakkynurenine levels are reached one day after LPS administration(Takikawa, O., et al. (1986) J. Biol. Chem. 261:3648-53; Yoshida, H., etal. (1998) Cell 94:739-750). The pharmacodynamic assay described here isbased on measuring serum levels of both kynurenine and tryptophan.Calculating the kynurenine/tryptophan ratio provides an estimate of IDOactivity that is independent of baseline tryptophan levels (Fuchs, D.,et al. (1991) Immunol. Lett. 28:207-11; Gasse, T., et al. (1994) Eur. J.Clin. Chem. Clin. Biochem. 32:685-9), and this approach to measuring IDOactivity has been used frequently in humans. The principle advantage ofthis approach over the direct assessment of IDO enzymatic activity intissue is that it is a non-invasive procedure permitting multiplesamples to be collected from the same animal. This enables IDO activityto be monitored in a single mouse at multiple time points. Tryptophanand kynurenine levels in the serum can be determined by HPLC analysis.The level of compound in serum can also be determined in the same HPLCrun, thus permitting concurrent collection of pharmacokinetic data in asingle experiment.

Methods:

FVB MMTV-neu male mice at ˜8-10 weeks of age can be used to perform thebulk of the pharmacodynamic analysis because their genetic background isthe same as that of the MMTV-neu females mice that are used in themammary gland tumor model to evaluate compound efficacy. Salmonellaminnesota mutant strain R-595 (S. minnesota R) derived LPS has beenshown to elicit the most sustained level of kynurenine induction in acomparative analysis of LPS preparations from different bacterialstrains (Yoshida, R., et al. (1981) Arch. Biochem. Biophys. 212:629-37).Because it provides the widest window in which to evaluate the impact ofIDO inhibitors, this preparation of LPS can be used in thepharmacodynamic assay. The minimum i.p. bolus dose of S. minnesota R LPSthat elicits maximal IDO activity has been reported to be ˜1 mg/kg andmaximal IDO activation is reached by ˜24 hr. following LPS treatment(Yoshida, R., et al. (1981) Arch. Biochem. Biophys. 212:629-37).

Serum levels of kynurenine and tryptophan can be quantitativelydetermined by HPLC analysis (Hwu, P., et al. (2000) J Immunol164:3596-9; Widner, B., et al. (1997) Clin. Chem. 43:2424-6; see FIGS.22A-D). By this procedure, serum concentrations of at least 1.25 μMkynurenine and 3 μM tryptophan are detected. In unchallenged FVB malemice the serum kynurenine is at or below the limit of detection andserum tryptophan is readily detectable at ˜50 μM (FIG. 22B). 24 hr afterLPS challenge, serum kynurenine is induced to ˜6 μM (FIG. 22C). 1MT inserum at a concentration of at least 5 μM can also be effectivelymeasured. This is well below the serum levels of ˜100 μM 1MT achievedwith biologically efficacious dosing of 2×140 mg 1MT pellets (FIG. 22D).

Compounds can be evaluated first by challenging with LPS and thensubsequently administering a bolus dose of compound at the time that theserum kynurenine level plateaus. The kynurenine pool is rapidly turnedover with a half-life in serum of less than 10 minutes (Bender, andMcCreanor (1982) Biochim. Biophys. Acta 717:56-60; Takikawa, O., et al.(1986) J. Biol. Chem. 261:3548-53) so that pre-existing kynurenine isnot expected to unduly mask the impact that IDO inhibition has onkynurenine production. The vehicle chosen for administration can dependin large part on the physical properties of each particular compound.The preferred vehicle is isotonic saline, but this requires that thecompound be soluble in aqueous solution. It is anticipated that somecompounds may not be sufficiently soluble, in which case the compoundscan be administered as a suspension in Methocel®/Tween® (0.5%methylcellulose/1% Tween® 80).

Each experiment can include non-LPS-exposed mice (to determine baselinekynurenine levels against which to compare the other mice) and a set ofLPS-exposed mice dosed with vehicle alone (to provide a positive controlfor IDO activation). Mice can be monitored following LPS administrationand immediately euthanized if they present with signs of pronouncedendotoxemia (ruffled fur, sluggishness). Each compound can initially beevaluated in mice at a single high i.p. bolus dose in the range of atleast 100 mg/kg. Blood can be collected at defined time intervals (forexample, 50 μl/sample at 5, 15, 30 min., 1, 2, 4, 6, 8, and 24 hr.following compound administration) for HPLC analysis of kynurenine andtryptophan levels (pharmacodynamic analysis) as well as for the level ofcompound (pharmacokinetic analysis). From the pharmacokinetic data thepeak serum concentration of compound achieved can be determined as wellas the estimated rate of clearance. By comparing the level of compoundin serum relative to the kynurenine/tryptophan ratio at various timepoints, the effective IC₅₀ for IDO inhibition in vivo can be roughlyestimated.

Based on the results of the single dose study, a second dose escalationstudy can be conducted for every efficacious compound. The study can be,for example, aimed at a maximum dose that achieves 100% IDO inhibitionat the peak concentration (if possible) in one cohort of mice and doseadditional cohorts with concentrations that decrease in 3-fold stepwiseincrements to cover a 2 log₁₀ range between the highest and lowestdoses. Accurate IC₅₀ determinations can be extracted from these data.The same approach can be used to test for oral bioavailability ofbiologically active compounds, first testing each compound at a singlemaximum concentration p.o. bolus dose and then further evaluating thosecompounds that exhibit significant oral efficacy in a dose escalationstudy. To ensure that in vivo responsiveness is not subject to sexualdimorphism, a single i.p bolus dose experiment can be carried out infemale mice at the calculated IC₅₀ dose for each active compound.

Example 10 Combinatorial Treatment of Tumors with an IDO Inhibitor and aSignal Transduction Inhibitor

The MMTVneu transgenic “oncomouse” model of breast cancer was used tomeasure the effects of IDO inhibitors and STIs on tumor pathophysiology.The MMTVneu transgenic mouse develops aggressive tumors of the mammarygland that resemble poorly differentiated human ductal carcinomas. Inthe MMTVneu mouse model, breast cancer is initiated by tissue-specificexpression of a mutant form of the HER-2/Neu gene that is activatedfrequently in aggressive human breast ductal carcinomas. HER-2 is amember of the EGF-R family of cell surface growth factor receptors. Mycis an obligate downstream effector for HER-2/Neu to drive cancer. FemaleMMTVneu “oncomice” are mated twice to initiate expression from the mousemammary tumor virus (wry) promoter which drives transcription of theNeu/HER2 oncogene in mammary tissue. Mammary tumors arise with apenetrance of >90% in this model system by 5 months of age. MMTVneu“oncomice” bearing similarly sized tumors of ˜150 mm³ were randomlyassigned to control or experimental treatment groups. Control mice wereimplanted with placebo time-release pellets (Innovative Research, Inc.,Sarasota, Fla.). Experimental groups of mice were (1) implanted with1MT-containing time-release pellets, (2) treated with L-744,832, or (3)implanted with 1MT-containing time-release pellets and treated withL-744,832. L-744,832, which mimics the CaaX motif to which the farnesylgroup is added, is a potent and selective inhibitor of farnesyltransferase (FTI) (Kohl et al., (1995) Nat. Med. 1(8):747-748).

The time-release pellets are composed of a copolymer which is inert andgradually dissolves and breaks down to a non-toxic substance thatremains largely localized during the course of the experiment.Time-release pellets impregnated with 1MT release a dose of 10 mg/dayfor a period of up to 14 days as documented by the commercial vendor(Innovative Research, Inc., Sarasota, Fla.). Two pellets per mouse wereimplanted to deliver a total dose of 20 mg/day. Therefore, for a 25 gmouse the total dose is 800 mg/kg/day or 280 mg over a 14 day period.Steady-state levels were reached within 12-24 hours and are maintainedthroughout the entire period based on the manufacturer's specifications.The delivered dose is effective at eliciting allogenic conceptusrejection (A. Muller, J. B. DuHadaway, G. C. Prendergast, unpublishedresults) as described by Munn et al. (Science 281:1191-1193, 1998).

Time-release pellets were introduced subcutaneously on the backs of miceanesthetized by intramuscular injection of ketamin/rompun. Bluntdissection with a hemostat is used to separate the skin from theunderlying muscle to create a subcutaneous pocket. One or twobiodegradable slow release pellets were implanted within this pocket,rather than directly under the incision in order to prevent mechanicalstress and wound dehiscence. The incision was then closed with woundclips. Based on the ability of female mice that have been implanted withplacebo time-release pellets to carry pregnancies to term, distress fromthe procedure appears to be negligible.

The signal transduction inhibitors, e.g., FTI L-744,832 were preparedand delivered to the mice as described in Kohl et al. (Nature Med.(1995) 1:792-797).

FIG. 23 summarizes the findings of the experiments to test the abilityof 1MT to cooperate with FTI L-744,832 (as well as chemotheraeuticagents) to cause regression of established tumors in MMTVneu “oncomouse”model. During the two week course of the experiment, an elevation of˜200% in the tumor volume of mock-treated control mice was observed.Treatment of mice with 20 mg/day 1MT, delivered by subcutaneoustime-release pellets, retarded but did not block tumor growth.Similarly, treatment of tumor-bearing mice with FTI L-744,832 retardedbut did not block tumor growth. In contrast, the combination of 1MT plusL-744,832 treatment caused tumor regression in the model.

Example 11 Assaying Novel IDO Inhibitors

A variety of compounds were screened for their efficacy as IDOinhibitors. Certain compounds were screened in a biochemical assay asfollows. IDO cDNAs were expressed in bacteria as his-tagged proteins andpurified as previously described (Littlejohn et al. (2000) Prot. Exp.Purif. 19:22-29). Briefly, the purified IDO was incubated with substrateand varying amounts of the IDO inhibitor candidate. The fluorescence ofthe reaction mixture was measured to determine the efficacy of thecandidate inhibitor because a product of the reaction, kynurenine, isfluorescent. The results of the in vitro biochemical screen are depictedin FIG. 24.

The candidate compounds were also screened in a cell-based assay (forsimilar assay see Munn et al. (1999) J. Exp. Med. 189:1363-1372).Briefly, human 293/Phoenix cells were transiently transfected with humanIDO or TDO cDNA expression vectors. The candidate compounds were addedto the transfected cells at various concentrations. Kynurenine wasquantitated in tissue culture media using a fluorescence-based protocol.The results from these experiments are presented in FIGS. 25-27.

As noted in these figures, the most potent inhibitors identified are aset of thiohydantoin derivatives of indoleamine. FIG. 26 providesresults using these particular inhibitors. The most potent of theseinhibitors, methyl-TH-DL-trp, displayed an inhibition of IDO activity2.7 times greater than 1MT at a concentration of 250 μM (FIG. 27).

In addition to the thiohydantoin derivatives of indoleamine, a group ofnatural products was screened. Interestingly, effective inhibitors fromthis group were compounds from foods with cancer preventitive properties(e.g. cruciferous vegetables). Brassinin, a compound found in Chinesecabbage, was scored as the most potent compound among the naturalproducts determined to be IDO inhibitors (FIG. 25A).

The toxicity of certain screened compounds was also examined. As seen inFIG. 28, most IDO inhibitory compounds are not intrinsically growthinhibitory or cytotoxic to neoplastically transformed breast or prostatecancer cells (FIG. 28).

Example 12 Combinatorial Treatment of Tumors with an IDO Inhibitor andCytotoxic Chemotherapeutic Agent

The MMTVneu transgenic “oncomouse” model of breast cancer was also usedto measure the effects of IDO inhibition and cytotoxic chemotherapeuticagents on tumor pathophysiology.

MMTVneu “oncomice” bearing similarly sized tumors of ˜150 mm³ wererandomly assigned to control or experimental treatment groups. Controlmice were implanted with placebo time-release pellets (InnovativeResearch, Inc., Sarasota, Fla.). Experimental groups of mice were (1)implanted with 1MT-containing time-release pellets as described inExample 10, (2) treated with paclitaxel) (Taxol®) or other cytotoxicagents, or (3) implanted with 1MT-containing time-release pellets andtreated with paclitaxel or other cytotoxic agents.

Time-release pellets were introduced subcutaneously on the backs of miceas described previously.

The cytotoxic chemotherapeutic agents were prepared and delivered to themice as follows. Paclitaxel was dissolved in equal volumes of absoluteethanol and the clinically-used solubilizing agent Cremophor® EL. Thesolution was sonicated up to 30 minutes and stored as a 20 mg/ml stocksolution at 4° C. for up to one week. Before use, this solution wasdiluted further at 1:5 with sterile physiological saline. Paclitaxelformulated in this manner was administered to mice by a single bolusintravenous (i.v.) injection into the tail vein. Mouse tails can bewarmed to facilitate identification and injection of the vein. Themaximum tolerated dose (MTD) of paclitaxel (13.3 mg/kg) was deliveredfive (5) times during the 2 week experiment on a thrice-weekly schedule(i.e., Friday—pellet implantation; Monday/Wednesday/Friday,Monday/Wednesday—paclitaxel inject; Friday—euthanize animals and harvesttumors for analysis). The MTD of cisplatin (1 mg/kg) was obtained as aclinical preparation in saline and delivered as a bolus i.v. injectionon the same schedule. Control treated mice received only the Cremophor°EL vehicle formulation without paclitaxel.

FIG. 29 and Table 1, in addition to FIG. 23, summarize the findings ofthe experiments to test the ability of 1MT to cooperate with twocytotoxic agents to cause regression of established tumors in MMTVneu“oncomouse” model. During the two week course of the experiment, anelevation of ˜200% in the tumor volume of mock-treated control mice wasobserved. Treatment of mice with 20 mg/day 1MT, delivered bysubcutaneous time-release pellets, retarded but did not block tumorgrowth. Similarly, treatment of tumor-bearing mice by intravenousinjection of paclitaxel or cisplatin at the maximum-tolerated dosesretarded but did not block tumor growth. In contrast, the combination of1MT plus paclitaxel or cisplatin treatment caused tumor regression inthe model. Similar results were observed with a reduction of paclitaxelto ˜25% the maximum-tolerated dose (data not shown). Inasmuch as thecytotoxic agents employed in these studies are known to be toxic to thevery T cells that the IDO inhibitors would allow to be recruited andactivated, these results are unexpected in view of the prior art.

TABLE 1 Un- 1MT Taxol 1MT + Cisplatin 1MT + treated only only Taxol onlyCisplatin Number of 5 5 5 6 3 3 Mice Number of 5 7 6 9 5 5 Tumors Mean195.1 80.27 139.4 −30.2 91.35 −27.94 Std. 97.54 73.12 118.1 30.7 118.535.1 Deviation Std. Error 43.62 27.64 48.2 10.23 53 15.7 Minimum 122.2 020 −78.4 −26.53 −67.86 25% 25 40.25 −56.5 Percentile Median 134.4 72.87130.4 −23.44 40 −30.56 75% 130.4 247.6 −6.445 Percentile Maximum 336.5215 360.8 12.5 255.6 14.29 Lower 73.95 12.65 15.52 −53.8 −55.79 −71.5295% CI Upper 316.2 147.9 263.3 −6.605 238.5 15.64 95% CI

Statistical analysis of the tumors of MMTVneu mice after varioustreatments. Numbers are provided as percent change in tumor volume ascompared to the tumor volume prior to treatment. Lower and upper 95% Clindicate lower and upper 95% confidence limits.

Histological and immunohistochemical analysis of tumor sections isolatedfrom the control and experimental cohorts revealed dramatic changes onlyin the tumor tissues from the mice treated with the combinatorialregiment. Most notably, evidence of pronounced hemorrhage, apoptosis,and infiltration of CD3-positive T cells was seen in the mice thatreceived the combinatorial regiment (data not shown). In conclusion, thecombined application of 1MT with cytotoxic agents was efficacious ineliciting regression of established breast tumors in the MMTVneu“oncomouse” model system.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

Several publications and patent documents are cited in the foregoingspecification in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

What is claimed is:
 1. A pharmaceutical composition comprising aneffective amount of a compound having indoleamine 2,3 dioxygenase (IDO)inhibitory activity and a pharmaceutically acceptable carrier medium,said compound being of formula (I):

wherein R₁ is H or lower alkyl; R₂ is H; R₃ is

wherein R_(A) is selected from the group consisting of (C₃-C₆)alkenyl,substituted or unsubstituted aralkyl and substituted or unsubstitutedcycloalkyl, and R_(B) is selected from the group consisting of H,(C₁-C₆) alkyl, (C₃-C₆) alkenyl, substituted or unsubstituted aralkyl andsubstituted or unsubstituted cycloalkyl, said substituted aralkyl andsubstituted cycloalyl having one or two substituents selected from thegroup of halo, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkythio,hydroxyl, carbonyl, epoxy, —C(═O)—OR, —OC(═O)—R, amino, —NHC(═O)—R,—NHCONH₂, —NHCONHR or thiol wherein R represents hydrogen or(C₁-C₄)alkyl; X, Y, and Z may be the same or different and are selectedfrom the group consisting of H, halogen, NO₂, and hydrocarbyl; with theproviso that formula (I) does not include3-(N-allyl-thiohydantoin)-indole.
 2. A pharmaceutical compositioncomprising an effective amount of at least one indoleamine2,3-dioxygenase (IDO) inhibitor and at least one signal transductioninhibitor (STI) in a pharmaceutically acceptable carrier medium, whereinsaid at least one IDO inhibitor is selected from the group of compoundsof the structure of formula (I):

wherein R₁ is H or lower alkyl; R₂ is H; R₃ is

wherein R_(A) is selected from the group consisting of (C₃-C₆)alkenyl,substituted or unsubstituted aralkyl and substituted or unsubstitutedcycloalkyl, and R_(B) is selected from the group consisting of H,(C₁-C₆) alkyl, (C₃-C₆) alkenyl, substituted or unsubstituted aralkyl andsubstituted or unsubstituted cycloalkyl, said substituted aralkyl andsubstituted cycloalyl having one or two substituents selected from thegroup of halo, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkythio,hydroxyl, carbonyl, epoxy, —C(═O)—OR, —OC(═O)—R, amino, —NHC(═O)—R,—NHCONH₂, —NHCONHR or thiol wherein R represents hydrogen or(C₁-C₄)alkyl; X, Y, and Z may be the same or different and are selectedfrom the group consisting of H, halogen, NO₂, and hydrocarbyl; with theproviso that formula (I) does not include3-(N-allyl-thiohydantoin)-indole.
 3. The pharmaceutical composition ofclaim 2, wherein said at least one STI is selected from the groupconsisting of bcr/abl kinase inhibitors, epidermal growth factor (EGF)receptor inhibitors, her-2/neu receptor inhibitors, farnesyl transferaseinhibitors (FTIs), inhibitors of Akt family kinases or the Akt pathway,and cell cycle kinase inhibitors.
 4. The pharmaceutical composition ofclaim 3, wherein said at least one STI is selected from the groupconsisting of STI 571, SSI-774, C225, ABX-EGF, trastuzumab, L-744,832,rapamycin, LY294002, flavopiridal, and UNC-01.
 5. The pharmaceuticalcomposition of claim 4, wherein said at least one STI is L-744,832.
 6. Apharmaceutical composition comprising an effective amount of at leastone indoleamine 2,3-dioxygenase (IDO) inhibitor and at least onechemotherapeutic agent in a pharmaceutically acceptable carrier medium,wherein said at least one IDO inhibitor is selected from the group ofcompounds of formula (I):

wherein R₁ is H or lower alkyl; R₂ is H; R₃ is

wherein R_(A) is selected from the group consisting of (C₃-C₆)alkenyl,substituted or unsubstituted aralkyl and substituted or unsubstitutedcycloalkyl, and R_(B) is selected from the group consisting of H,(C₁-C₆) alkyl, (C₃-C₆) alkenyl, substituted or unsubstituted aralkyl andsubstituted or unsubstituted cycloalkyl, said substituted aralkyl andsubstituted cycloalyl having one or two substituents selected from thegroup of halo, (C₁-C₄)haloalkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkythio,hydroxyl, carbonyl, epoxy, —C(═O)—OR, —OC(═O)—R, amino, —NHC(═O)—R,—NHCONH₂, —NHCONHR or thiol wherein R represents hydrogen or(C₁-C₄)alkyl; X, Y, and Z may be the same or different and are selectedfrom the group consisting of H, halogen, NO₂, and hydrocarbyl; with theproviso that formula (I) does not include3-(N-allyl-thiohydantoin)-indole.
 7. The composition of claim 6, whereinsaid at least one chemotherapeutic agent is selected from the groupconsisting of paclitaxel (Taxol®), cisplatin, docetaxol, carboplatin,vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11,5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin(doxorubicin), etoposide, arsenic trioxide, irinotecan, and epothilonederivatives.
 8. The pharmaceutical composition of claim 6, wherein saidat least one chemotherapeutic agent is paclitaxel.